Prolonged ischemia with or without reperfusion triggers the apoptotic and non-apoptotic death of cardiac myocytes. The death of these cells is a major component in the pathogenesis of myocardial infarction. Apoptosis and some non-apoptotic forms of cell death are actively mediated processes, suggesting that they can be inhibited to therapeutic advantage in heart disease. We and others have shown that inhibition of cardiac myocyte death during ischemia-reperfusion in the intact mouse limits infarct size and preserves cardiac function. Apoptosis is mediated by two central pathways, both of which play important roles in cardiac myocyte death during ischemia-reperfusion. ARC is an endogenous inhibitor of apoptosis that is abundant in cardiac myocytes. ARC is unique among apoptosis inhibitors in that it antagonizes both central apoptosis pathways through mechanisms that we have elucidated. ARC also inhibits necrotic cell death through unknown means. Given the abundance and potency of ARC, why do large numbers of cardiac myocytes die during ischemia- reperfusion? A possibility is suggested by the dramatic decreases in ARC protein levels during ischemia- reperfusion. We have shown that these decreases are the trigger - not the consequence - of the resulting cell death because maintenance of ARC even at sub-baseline levels inhibits cell death. We have determined that ARC protein levels decrease during ischemia-reperfusion primarily because of increases in ARC protein degradation, and that ARC protein is degraded by both the ubiquitin-proteasomal pathway and in the lysosome via autophagy. The objective of this project is to define the molecular mechanisms that mediate ARC degradation during ischemia-reperfusion in vivo and to determine the contribution of each to the loss of ARC protein and resulting cardiac myocyte death. Aim 1 investigates the ubiquitin-proteasomal pathway in this process. In particular, the mechanisms by which the E3 ligase MDM2 act on ARC will be delineated, as will the potential role of a novel phosphodegron in ARC that we have identified. Aim 2 focuses on the lysosomal pathway and seeks to identify the form(s) of autophagy that mediate ARC degradation. Of note, we have identified a novel motif in ARC that may target ARC for chaperone-mediated autophagy. Aim 3 tests the mechanistic findings from the earlier aims in mice genetically engineered to have defects in key protein degradation mechanisms. The roles of these mechanisms in ARC degradation and the genesis of myocardial infarction in vivo will be delineated. These studies will provide novel insights into the events that trigger cardiac myocyte death during myocardial infarction. In addition, they may provide the conceptual basis for the identification of targets for small molecule therapies to maintain ARC levels during myocardial infarction so as to (a) limit infarct size and (b) expand the time window for reperfusion therapy with primary angioplasty. PUBLIC HEALTH RELEVANCE: ARC is a protein that protects heart muscle cells from dying, but at the beginning of a heart attack ARC is destroyed allowing heart muscle cells to die. The purpose of this research is to determine why and how ARC gets destroyed. The resulting knowledge may be useful in devising a medication to prevent ARC destruction and lessen the damage from heart attacks.